Expression plasmids and production of EGFP in stably transfected Acanthamoeba.

Expression plasmids and production of EGFP in stably transfected Acanthamoeba.
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DOI:
10.1016/j.pep.2009.10.008
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发表时间:
2010-03
影响因子:
1.6
通讯作者:
Bateman, Erik
Bateman, Erik
中科院分区:
生物学4区
文献类型:
--
作者:
Bateman, Erik

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New plasmids containing the TATA-Binding Protein (TBP), TBP Promoter Binding Factor (TPBF) or Glyceraldehyde Phosphate Dehydrogenase (GAPDH) gene promoters from Acanthamoeba castellanii are described. The promoters for Acanthamoeba TPBF and GAPDH genes were used to drive constitutive expression of enhanced green fluorescent protein (EGFP) in stably transfected Acanthamoeba. Based initially on fluorescence microscopy and SDS PAGE analysis of EGFP, both promoters produce robust expression of EGFP, with the highest level obtained from the GAPDH gene promoter in cells grown in low concentrations of neomycin G418. Purification of EGFP from lysates of 22-ml cultures by conventional chromatography yielded approximately 1.1 milligrams of EGFP, a value that extrapolates to 50 milligrams per liter of cell culture. The results suggest that Acanthamoeba is a useful cost-effective system for the production of recombinant proteins.
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